Estimation of paracellular conductance of primary rat alveolar epithelial cell monolayers

Freshly isolated rat type II pneumocytes, when grown on permeable tissue culture-treated polycarbonate filters, form confluent alveolar epithelial cell monolayers (RAECM). Cells in RAECM undergo transdifferentiation, exhibiting over time morphological and phenotypic characteristics of type I pneumocytes in vivo. We recently reported that transforming growth factor-β1 (TGF-β1) decreases overall monolayer resistance ( Rte) and stimulates short-circuit current in a dose-dependent manner. In this study, we investigated the effects of TGF-β1 (50 pM) or 10% newborn bovine serum (NBS) on modulation of paracellular passive ion conductance and its contribution to total passive ion conductance across RAECM. On days 5–7 in culture, tight-junctional resistance ( Rtj, kΩcm2) of RAECM, cultured in minimally defined serum-free medium (MDSF) with or without TGF-β1 or NBS, was estimated from the relationship between observed transmonolayer voltage and resistance after addition of gramicidin D to apical potassium isethionate Ringer solution under open-circuit conditions. NaCl Ringer solution bathed the basolateral side throughout the experimental period. Results showed that transmonolayer conductance (1/ Rte) and tight-junctional conductance (1/ Rtj) are 0.59 and 0.14 mS/cm2 for control monolayers in MDSF, 1.59 and 0.38 mS/cm2 for monolayers exposed to TGF-β1, and 0.38 and 0.18 mS/cm2 for monolayers grown in the presence of NBS. The contributions to total transepithelial ion conductance by the paracellular pathway are estimated to be 23, 23, and 47% for control, TGF-β1-exposed, and newborn bovine serum (NBS)-treated RAECM, respectively.

Maintenance of bile acid synthesis and cholesterol 7α-hydroxylase activity in cultured rat hepatocytes

Addition of foetal-bovine serum to rat hepatocytes cultured in Williams E medium resulted in improved maintenance of bile-acid-synthetic capacity and cholesterol 7 alpha-hydroxylase activity as compared with cultures supplemented with rat or newborn-bovine serum or cultures in a hormonally defined serum-free medium. Minimally, 5% (v/v) foetal-bovine serum was necessary to maintain these liver-specific functions. Serum factor(s) responsible for these effects were not dialysable or associated with lipoproteins, but were removed by charcoal extraction.

Acellularity of starfish embryonic mesenchyme cells as shown in vitro

Embryonic mesenchyme cells of the starfish are shown to be unexpectedly fusogenic in vitro. When archenteron complexes (archenterons and varying portions of the extracellular matrix {ecm} surrounding them) are isolated from starfish embryos and inoculated in sea water containing 4% newborn bovine serum, the mesenchyme cells form large syncytia on the substratum underneath each archenteron. These syncytia break into smaller fragments interconnected by fine cell processes within 24h. These networks have been studied morphologically, dynamically and ultrastructurally and found to lack cell borders between the constituent fragments. These fragments contain various numbers of nuclei ranging from 0 to 6 or more and move about constantly over the substratum, sometimes breaking into two and sometimes fusing with neighbouring fragments, so that the overall pattern of the network changes constantly. Our results also indicate that the network is three-dimensional i.e. it has crossing sites, the frequency of which seems to depend on the amount of the ECM excreted on the substratum. A similar network pattern is found among mesenchyme cells in vivo, which suggests that the features found in vitro reflect those in vivo.